1. Technical Field
The present invention relates to a novel in vivo antigen which can serve as a diagnostic marker for sepsis. The invention also relates to a method for diagnosing the sepsis characterized by assaying the antigen, and an assay kit and an assay method for the antigen using a particular antibody. The invention further relates to a recombinant soluble fragment useful as a standard substance of the assay kit, an antibody binding to the fragment, a method for producing the fragment, and an antibody screening method using the fragment.
2. Background Art
A CD14 molecule was named as a protein identified by a family of antibodies that recognize glycoproteins expressed on the membrane surface of monocytes in Third Leukocyte Typing Conference, 1986. In 1990, Wright et al. elucidated that the CD14 molecule is a receptor for LPS, endotoxin (“Science”, vol. 249, p. 1431-1433, 1990). The CD14 molecule is a glycoprotein having a molecular weight of 53-55 kDa, and analyses on cDNA revealed that mRNA of about 1.4 kb has coding sequence of 356 amino acids (“Nucleic Acids Research” (U.K.), vol. 16, p. 4173, 1988).
It was reported that human CD14 molecules include soluble CD14 molecules in addition to membrane-bound CD14 molecules and blood contains soluble CD14 molecules having different molecular weights (“European Journal of Immunology” (Germany), vol. 23, p. 2144-2151, 1993). In addition, Landmann et al. conducted Western blot analyses on soluble CD14 in serum of patients suffering from sepsis and reported that soluble CD14 of about 55 kDa is at high levels in non-survival sepsis patients and patients with paroxysmal nocturnal hemoglobinuria (PNH) and that in normal sera, this molecule was not detected but soluble CD14 of 49-kDa, a slightly lower molecular weight than the former, was detected (“The Journal of Infectious Disease” (U.S.A.), vol. 171, p. 639-644, 1995).
Stelter et al. reported that the difference in sugar chains is involved in those subtypes having different molecular weights and two soluble CD14 subtypes having different molecular weights are found in blood even after removal of N- and O-linked sugar chains (“European Journal of Biochemistry” (Germany), vol. 236, p. 457-464, 1996). In addition, Bufler et al. conducted the C-terminal analysis on soluble CD14 and reported that a GPI anchored to a serine residue at position 327 of soluble CD14 and that a soluble CD14 molecule having a molecular weight of about 56 kDa is one of the molecular species from which GPI is not anchored (“European Journal of Immunology” (Germany), vol. 25, p. 604-610, 1995).
Concerning recombinant full length soluble CD14 and a fragment thereof, Juan et al. reported that an N-terminal 1-152 fragment of human CD14 has a function of transmitting LPS signals to cells (“Journal of Biological Chemistry” (U.S.A.), vol. 278, p. 1382-1387, 1995), but they have not succeeded in expressing an N-terminal 1-124 fragment and an N-terminal 1-98 fragment. In addition, Majerle et al. reported that an N-terminal 1-152 fragment of human CD14 which includes 3 units of a leucine-rich repeat (LRR) region is refolded and an N-terminal 1-134 fragment of human CD14 which includes only two units of the LRR region is not refolded, and that they did not succeeded in expressing an N-terminal 1-69 fragment (“Pflugers Arch-European Journal of Physiology” (Germany), vol. 439 [Suppl], p. R109-R110, 2000), but according to the report, they have not succeeded in expressing an N-terminal 1-69 fragment.
Antibodies against CD14 molecules include many anti-CD14 antibodies, which have been prepared and used in identification of CD14 proteins, such as MEM-18 prepared by Bazil et al. (“European Journal of Immunology” (Germany), vol. 16, p. 1583-1589, 1986), RoMo-1 prepared by Shutt et al. (“Allergie und Immunologie” (Germany), vol. 34, p. 17-26, 1988), and 3C10 prepared by Steinman et al. (“Journal of Experimental Medicine” (U.S.A.), vol. 158, p. 126-145, 1983).
Furthermore, soluble-CD14 assay systems using those antibodies have been reported by Shutt et al. (DE-286876-A), Bazil et al. (“Molecular Immunology” (U.K.), vol. 26, p. 657-662, 1989), and Grunwald et al. (“Journal of Immunological Methods” (Holland), vol. 155, p. 225-232, 1992), allowing the assay of soluble CD14 in human body fluid.
Furthermore, soluble CD14-ELISA kits have been released on the market from IBL-Hamburg, Medgenix, and R & D Systems, and the assay of soluble CD14 has been performed for many diseases such as sepsis (“Clinical Immunology And Immunopathology” (U.S.A.), vol. 80, p. 307-310, 1996; and “Rinshokensa”, vol. 38, p. 341-344, 1994).
However, it was found that soluble CD14 is not a sepsis-specific marker because of increases in levels of soluble CD14 molecules of about 55 kDa and 49 kDa (from report to report, the molecular weights are different and not limited to about 55 kDa and 49 ka, and the same will be applied in the following description) depending on the degree of progress of diseases even in diseases except sepsis (“Infection and Immunity” (U.S.A.), vol. 67, p. 417-420, 1999; “Clinical and Experimental Immunology” (U.K.), vol. 120, p. 483-487, 2000; and “Clinical Experimental Immunology” (U.K.), vol. 96, p. 15-19, 1994). Furthermore, the soluble CD14 was expected to be a marker for the severity of sepsis. However, the soluble CD14 has not been provided as a diagnostic product for sepsis because of no correlation with septic shock (“Pediatric allergy and immunology) (Denmark), vol. 8, p. 194-199, 1997) and also no correlation with systemic inflammatory response syndrome (SIRS) (“European Journal of Clinical Investigation” (U.K.), vol. 28, p. 672-678, 1998).
Further, there has been found out the presence of a soluble CD14 molecule with a low molecular weight of about 36 kDa in blood in addition to others such as two kinds of soluble CD14 molecules described above of about 55 kDa and 49 kDa reported by Landmann et al. (high molecular weight CD14 (from report to report, the molecular weights are different and not limited to about 55 kDa and 49 ka, and the same will be applied in the following description). There has been also found out the presence of a small amount of the low-molecular-weight CD14 in a normal donor and of an increased amount of the low-molecular-weight CD14 in patients suffering from sepsis. Consequently, the clinical efficacy of the assay on a soluble low-molecular-weight CD14 has been validated. As an assay for the soluble low-molecular-weight CD14, there is a proposal in which the level of low-molecular-weight CD14 in blood is indirectly obtained by subtracting the level of high molecular weight CD14 in blood from the total level of the soluble CD14 in blood (International publication WO 01/22085).